Reconstruction of human maxillary defects with nacre powder: histological evidence for bone regeneration

% Acadkmie des sciences / Elsevler. Paris Sciences m6dicales / Medical sciences

Reconstruction of human maxillary defects with nacre powder: histological evidence for bone regeneration Reconstruction de /‘OS alv@olaire maxillaire humain par de la poudre de nacre : preuve histologique d’une t+g&+t-ation osseuse G&CARD ATMN’,

NICOLE

BALMAIN’,

SOPHIE

BERLAND~,

BERNADETTE

VIDAL’,

I?VELYNE

LOPEZ~*

Huit patients porteurs d’un d&it osseux alvkolaire maxillaire dans les secteurs premolomolaires ont bkYici& d’une reconstruction par de la poudre de nacre provenant de la coquille de I’huitre g&ante Pinctada maxima. Au 6’ mois postopkatoire, des etudes histologiques, en lumit-re polarike et microradiographiques, rCalisCes sur des prelt-vements biopsiques montrent une parfaire osttointigration de la nacre qui est ktroitement soudCe B 1’0s nouveau qu’elle g&&e. La nacre subir une biodissolution cent-rip&e progressive et est remplacee par de 1’0s n&oformk d’abord immature puis lamellaire, mature. L’objectif kait d’obtenir une restauration osseuse de la crire alvColaire maxillaire. Cette etude conforte les donnks obcenues anttrieurement in vitro. Mats

cl&

: OS chkolaire,

dkfauf

osseux,

nacre,

osGogen&e,

biomatkriei

AEETRACl The

dejhive

reconstructed

ayeas using

in

the premoh-molar

powdered

graphic and polarized light studies was tightly bound to newly-formed replaced with immature and then previous Key words:

VERSION

experimental alveolar

in virro bone,

data bone

of maxillay

region

nacrefiom

thegiant

oyster

Pinctada

of drill

alveolar

bone

maxima.

of eight

Histological,

patients

were

mirrosadio-

biopsies taken 6 months b one. The nacre was gradually mature Iamellar bone. The

postoperativeb showed that ~zacre and ce~ltripetarj hiodissolved afld results are ill agreement with our

indicating

osteogenic

defect,

that nacre,

Ilaue

has good

osteogenesis,

properties.

biomaterial

ABREGEE

Ni les greffons osseux autologues ni les biomateriaux n’ont apportb B ce jour de r&hats parfaits dans la reconstruction des pert-es de substance osseuse. La nacre, qui forme la partie

C. R. Acad. Sci Paris, Sciences 1997. 320,253.258

de la vie / Life Sciences

inrerne de la coquille de I’hultre gCante Pinctada maxima, a ktP implant&e chez le chien, le moulon et le rat. Nous avons montre qu’une nCoformation osseuse intervient, confortant

253

G Atlan

et al.

les rkwltats obtenus pr&&demment in vitro. Ces resultats confirment que la nacre stimule l’ost&ogenkse et induit une r&$&ration osseuse. Huit patients (femmes de 48-55 ans) ont CtC retenus, porteurs d’une Cdentation partielle primolomolaire maxillaire avec une perte importante d’os alveolaire. De la poudre de nacre a et6 implantte en situation sous-gingivopCriostPe. Aprks 6 mois, des biopsies par carotage sont effect&es et soumises B une &valuation histologique, en microscopic photonique et en lumitre polarike et par microradiographie. La poudre de nacre est progressivement biodissoute de faGon centripkte et remplacke par de 1’0s ntoformk Les interfaces nacre-os neoformC montrent une soudure Ctroite, sans interposition de rissu mou ou de tissu fibreux. La nacre est graduellement remplacee par de 1’0s fibreux, immature, puis secondairement par de 1’0s lamellaire, mature, prtsentant une orientation kpondant aux contraintes mkaniques. Un tissu vasculaire, carackistique de la moelle osseuse, colonise progressivement les lacunes de 1’0s spongieux nkoform& L’os autologue est le seul biomatkriau connu pour induire une stimulation des cellules prkurseurs des ostkoblastes et provoquer une nkoformation osseuse. Cependant, le prtkvement de greffons osseux n’est pas saris risques et la quantitk d’os disponible reste limitee. De nombreux efforts sont entrepris pour produire et dCvelopper de nouveaux matkriaux implantables. La nacre est actuellement ktudiee comme un biomatkriau utilisable en chirurgie et en reparation osseuse. Dans cette kude, effect&e au 6’ mois postopkratoire, la ntoformation osseuse a atteint la partie centrale de la nacre implant&e. A la lumittre des rksultats que nous avons d@ obtenus in vivo et in vitro, nous suggkons que la nacre stimule. au temps initial, les cellules osteoprogtnitrices, les preostioblastes et les ostioblastes. Le biomattriau nacre joue le rBle de support et lib&e aussi des facteurs actifs au tours de sa biodissolution. Les cellules environnantes participent pr& cocement B la dkgradation de la nacre. Ces cellules dissolvent d’abord la partie minerale, lib&ant la matrice organique de la nacre qui est supposCe presenter des propriMs ostCog&niques. Les facteurs actifs de la nacre permettraient une nkoformation osseuse prkoce, suivie d’une activite ostioblastique continue telle que celle observke B 6 mois post-opkatoire. Nous suggkrons que les cellules formatrices d’os, ainsi stimul&es par la nacre, s&r&tent des facteurs locaux qui favorisent la formation continue d’os nouveau, regulant de faGon autocrine et paracrine son dPveloppement et permettant la mise en place d’un OS mature, physiologiquement sain.

bones 131. Autologous bone grafts are by far the most widely used material for bone reconstruction. Allogenic banked bone has also been employed [S, 61 but neither of these two solutions provides ideal results. The bone substitute used for implants should ideally have biological properties similar or identical to those of natural bone, and perhaps the most important of these is the capacity to initiate osteogenesis. Powdered nacre (mother-of-pearl), obtained from the inner shell layer of the giant oyster Pinctada maxima, has been implanted in bone of dog, sheep and rat with good evidence of local osteogenic activity and osteointegration with bone host [7-g]. Our previous in vitro studies have shown that nacre induces cultured human osteoblasts to form bone I1 0, 1 II. Thus, nacre contains one or more constituents that stimulate bone regeneration without any apparent cytotoxicity [I 0, 11 I. This study evaluates the suitability of nacre powder as an implant for the reconstruction and regeneration of maxillary alveolar ridge bone in human.

Materials

Reconstructive surgery patients, aged between upper edentation in the losses of alveolar ridge implants under optimal Nacre

powder

A number of biomaterials have been used in the reconstruction of facial bone defects and it has been shown that none of the biomaterials tested to date have been entirely successful [l-4]. Furthermore, it has been demonstrated that the reconstruction of the cranial and maxillary bones requires the incorporation of material with properties different from those required for the reconstruction of long

254

was performed on eight female 48 and 55 years old, with partial premolo-molar area and massive bone, which precluded dental conditions.

preparation

for

implantation

The nacre powder was obtained from the inner shell layer of the giant oyster Pinctada maxima. The powder (particle size: 50-100 pm), prepared by Centre de Transfert de Technologie Gramique ICTTC, Limoges, France), was mainly inorganic material, calcium carbonate, as aragonite crystals, plus 1.7% organic material (fibrous and nonfibrous proteins) (12, 131. It was sterilized by irradiation using the dose [gamma rays, 2.5 Mrad) recommended for sterilizing bone for bone banks, which has no deleterious effect on the osteogenic properties of the bone matrix [I 4, 151. The nacre powder was mixed with the patients own blood (autologous venous blood) under sterile conditions immediately before surgery. This provided a coagulum that was readily manipulated, moulded and adhered to the underlying host bone. Implantation

Introduction

and methods

site

All surgery was performed under local anaesthesia without any prophylactic treatment. The implant site was undermined via a curved fibromucosal incision and an elongated tissue expander was inserted and gradually filled with saline. The expander was removed and nacre powder inserted, under pressure, into the tunnel using a syringe so that it was in close contact with the alveolar bone. The wound was then closed and sutured with a monofilament. C. R. Acad.

Sci

Paw

Sciences

de la vie / Life Sciences 1997. 320,253-258

Nacre Clinical

follow-up

Clinical

follow-up

no infection showed an

and

biopsy

indicated

and X-rays X-ray-opaque

of the alveolar The endo-osseous reconstructed

rapid

mucosal

healing

with

taken at 6 weeks postoperatively tissue that increased the height

found in the

close newly

(figure

~AI.

to the diameter

mucosa trephine.

were

taken

at

Examination that woven

and

biopsies

microradiographic

were

procedures

immediately

fixed

in 70%

ethanol

embedded, undecalcified, in methylmetacrylate. (I 00 pm) were prepared using an lsomet microtome hler, III, USA) fitted with a low speed diamond undecalcified 15-30 pm, buffered polarized

tact

microradiographs

at pH 2.3 light with

and examined DMRB (Leicaj were

grinding (100 X-ray apparatus

exposures

and

Sections (Buesaw. The

sections were then ground to a thickness stained with PAS, basic fuschin or Toluidine

blue and before 1008

at 30 kV

of

under transmitted microscope. Con-

obtained

of

each

section,

pmj, with a Massiot-Philips using a Kw. copper target,

PW with

20 mA

film.

and

on

Kodak

HR

SO-343

(figure a degree

Low power histology (figure (figure 1 B) of the total area no

evidence

There were was in close

sue

the

reaction

or

showed of the

new nacre

persistent implant but the with the newly formed bone cancellous showed

between

islets (very less dense newly

newly

interfaces

med bone or fibrous

formed

between

were a bone tissue (figure

nacre

were

length porated

of the surfaces. isolated areas

tightly

that the nacre formed woven

bone

the bond, IC,

nacre without E). Thus,

welded

dense formed

The of

and

nacre

and

the

(proteoglycansi, They were

present lacunae

bone

at the

osteocytes flat lacunae.

in the The

penetrated

by hematopoietic

A

successful

newly residual

for-

along

formed nacre

the

soft the

whole

bone had implant,

incorwhich

islets appeared bone (figure

to be tightly linked by newly 1 F and 28~. The bone surfaces

lined

formation

Sci 1997. 320.253258

a component with osteoblasts, (figure

1 C and

Paris, Sciences

of 2Ai.

that stained metachromatic of acidic

purple with proteins

bone organic matrix. indicating continuous Osteoclasts

de la vie / Life Sciences

were

not

indicated implanted

The microradiographs similar to that of host

periphery

of the

lamellar newly

bone formed

newly

formed

were in well-orientated cancellous bone

tissue

(figure

bone

graft

requires

1 A, C and

both

osteogenic processes. Thus, help activate the local cell

problems with one is limited

~AJ, The was 2Aj.

sequently,

efforts

alloys, or most The main of ceramics

tage such

of suitable materials as ceramics to them.

material for bone 271. However, the be difficult to take

Pi,lctada

bone bank

maxima,

curand

have

been

The are

one

for and are main not

a fibrous union [I 8, 191. Conmade

over

All

the

[I 8, 191 materials, derivatives, suffer from

is a lack of biocompatibility, and aluminium alloys cause

and cannot be used clinically 1221. natural materials such as coral [181 but and are mechanically poor. This shorhas led to the use of composites reproduce natural systems and

Organic-metal

implants the still

increased appears

have

recently

use of to be the

new best

replacement and/or regeneration [24availability of bone is limited and it can samples from the iliac crests of patients

from diseases of contamination necessary lost bone forms the

material increase

material 115-171

grafts. allografts

polymers, carbon recently ceramics.

been used 1231. Thus, despite materials in recent years, bone

it is replace which

to

or so to develop inorganic natural [4, 20, 211 artificial implantable

osteomalacia are a few are resorbed

suffering is a risk

is the only properties

into the host site, so that restricts bone regeneration

considerable

on

Bone these

using autogenous availability. Bone

local There they

improve

has

osteoconductive

an implanted population

grafts are the most effective materials regeneration in cranial, maxillary surgery 1.31. Kevertheless, there

well incorporated is formed which

shortcomings. but implants newly

light the

Discussion

mostly metals, hydroxyapatite,

X-raysj bony tis-

polarized throughout

randomly distributed lacunae (figure 1 E and is characteristic of rapidly formed woven bone.

to

any intervening the bone and

together

were covered with osteoid borders with basic fuschin (figure IA, C) or Toluidine blue indicating the presence

C R. Acad.

large, which

past 10 years or synthetic

seemed to gradually disappear (figure 1 C, Ej. Examination of undecalcified sections under polarized light indicated

bone

trabeculae

network areas with

1 B).

Interfaces All

of

a consistent microradiographs

nacre implant radiologically

(figure

inflammatory

The serial sections the whole depth

areas contact

trabeculae, forming (figure 1 A, B). The residual between

The

autogenous bone producing bone facial reconstructive

1 A) and microradiography of the biopsies

of any

reaction to a foreign body. bone formation throughout implant. material

formed

1 F and 2B). of mineralization

osteoblast development. rently available that

was

while they were in Howship’s

woven bone were covered with lamellar bone with a stress-strain orientation [figure 1 F and 2B). The newly formed bone trabeculae contained many osteocytes lying in

and must

Results

There

newly

under transmittecl bone was formed

material showed bone.

The

of the

regeneration

bone

ridge

Drill biopsies perpendicular this time using a 3-4 mm Histological

maxillary

to the nacre implant, formed bone, located

Characteristics

by 3-4 mm and its width by 4-8 mm. dental implants were inserted into the maxillary ridge 6 months after rebuilding.

and

such as myopathies. Lastly, there in bone bank material. Hence,

to find a bone substitute that will and stimulate bone regeneration. nacre, inner part of the shell of the giant oyster is presently

being

tested

for

its perfor-

255

G. Atlan

et al.

figure

1. fffects

Histological nacre (NJ Correlation throughout are covered

of powdered

implant

on bone

regeneration

iB, C), and polarized bone (6). 6dsic fuchsi/l

in human light staining

maxillary

defects.

!F) examinatjon iA, c, /!1.

of undecalcified

sections

oi bone

biopsies

6 months

after

between histology iA/ and microrad/ography iBJ of the same section shows cancellou new/y formed bone (bl ilow X-rays densityj the nacre !h’i Implant (high X-rays densityi. They are tfghtly welded together, without Intervening fibrous tissue iC, El. Bone surfaces with osteoid borders ‘C, empty arrowssl lined with osteobla5tsCC, thin arrows). Po/drized I/ght in mdicates that bone trabeculae (E)

are made oi woven 15 x 2721.

256

nacre

(A, C, El, microradiograph/r imp/antatiOn. New/y formed

bone

iwl

in c-he

contact

wth

nd(.re

imphnt,

rovered

with

iamellar

bone

i/i

C. R. Acad.

‘final

magniiication

SCI. Paris,

Sciences

A, B: x 44;

B, C: x 130; D,

de la vie / Life Sciences 1997. 320,253.258

Nacre

figure

2. Newly

formed

bone

Histology surfaces

(Ai and polarization covered with osteoid

lacunae

(headarrows)

actively

surfaces

and bone

new-iormed

bone

(fina/

mdgnditation

mance as a surgical bone repair material in humans. We have shown that nacre has good osteogenic properties in vitro [lo, 111. It induces bone formation when placed close to cultured human osteoblasts, without addition of any of the chemical inducers usually required for in vitro bone formation. We have also used nacre powder in vivo for bone reconstruction in animals [7-g]. The present study on defects in human alveolar maxillary bone implanted with nacre shows that new bone formation extended deep into the nacre implant in all patients. The powder provides optimal conditions for the inward vascularization, growth of cells and bone formation. Our earlier in vitro studies [I 0, 1 I] showed that osteoblasts are attracted to the implant material, which acts as a carrier. The results of the present study suggest that the preosteoblasts and osteoblasts attracted to the nacre are also involved in the dissolution of the implant. nacre is gradually removed. This is corroborated by other studies that indicate that the cells in close contact with nacre, subcutaneously in rats, release collagenase 1281. However, no Trap-resistant C. R. Acad. Sci. Paris, 1997. 320,253-258

Sciences

maxillary

bone

regeneration

bone;

/. ldndhr

bone.

cells.

ill). &SIC fuchsIn itairing iA), N. nacre imphnt.; b: r~wi; io~med borders iempty drlow>l iined wfth osteob/a5ts /fh~n arrows), the boi~r

resorbing

and

de la vie / Life Sciences

bone,

ioiu~ing

\I’. ivoven

cc/h,

Bone

o~teoc idsts, hc dted in ~owsh~ps

x ,701.

cells, indicating ted.

the lack of active osteoclasts,

were detec-

The present data demonstrate that the tissue observed at the interface between nacre implant and newly formed bone consists of bone and nacre, corroborating and extending our previous findings obtained in vitro [lo, 111. The osteogenic activity of nacre is believed to be due to components of its organic matrix, but the specific agents have not yet been identified. These agents could be responsible for the cellular and osteoblastic activation leading to active osteogenesis. This osteogenesis produces a physiological cancellous bone. This study shows that woven bone is rapidly formed in close contact with nacre implant and then lamellar bone, as is usual during the ingrowth and development of bone 129, 301. We believe that bone progenitor cells are stimulated by nacre to produce autocrine or paracrine growth factors that act in the stimulation of osteogenesis. Studies are currently in progress to identify these factors and par-

257

G. Atlan

et al

titularly

those

of nacre

18, 9, 281.

contained

in hydrosoluble

organic

matrix

Conclusion The

eight

were

given

patients nacre

Acknowledgments:

suffering

from

implants.

The

authors

maxillary

Histological

thank

ridge and

Dr Owen

bone

loss

microradio-

Parkes

for

critical

graphic

examination

showed

that

had

induced

ding

of the

of drill

nacre

biopsies

implants

the formation deficient

perfectly

of new

maxillary

stimulate for the

elements

bone

reading

of the

in new

english

bone

ridge.

nacre implants may both and acts as a scaffolding involved

taken

were

6 months

later

tolerated

and

leading

We

to rebuil-

demonstrate

new laying

bone down

that

formation of various

reconstruction.

manuscript

and

Delphine

Vinet

for

the

photographs.

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